PROJECT SUMMARY Calcific aortic valve disease (CAVD) is a prevalent condition for which the only approved treatment is surgical valve replacement. Age and male sex are the dominant risk factors for developing this disease, which is characterized in its earliest stages by a change in extracellular matrix (ECM) composition. Moreover, recent evidence has indicated that the pathogenesis of CAVD in men may differ from that in women. We hypothesize that male and female valvular cells exhibit differential responsiveness to their microenvironment (i.e., ECM) and pathological cues (i.e., co-morbidities or risk factors), resulting in divergent disease trajectories for men and women. While clinical and animal studies can provide snapshots of disease progression, they do not permit the tailorability and high throughput needed to investigate this question and precisely monitor the specific cell/microenvironment/stimulus interactions that may yield mechanistic information about disease progression. Thus, we propose to develop in vitro disease-mimicking environments. Specifically, we will use novel tissue engineering and ECM-editing methods to create 3D engineered environments that recapitulate key features of the valve ECM and valvular interstitial cell phenotype at various stages of CAVD, and then use these scaffolds/tissues to examine disease progression upon exposure to risk factors and pathological stimuli. These defined, controlled environments will enable hypothesis testing that is impossible in humans and very difficult or not practical in animals, and will allow a prospective etiological investigation of sex bias in events that contribute to CAVD through execution of the following Aims: Aim 1: Construct engineered models that represent the valve microenvironment at different stages of disease. Aim 2: Investigate sex bias in the onset of inflammation using engineered models of CAVD. Aim 3: Investigate sex bias in the progression of fibrosis using engineered models of CAVD. Aim 4: Design organ cultures to validate engineered disease models and probe mechanism. This work has the potential to identify specific valve changes that can inform the design of sex- or stage- specific intervention strategies to reduce CAVD risk or progression and sets the stage for future work to improve CAVD prevention and treatment. We will make these advancements through our implementation of transformative approaches and tools to study ECM (patho)biology, which can also be readily applied to other cardiovascular (or non-cardiovascular tissues).